The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulated expression of viral genetic information. Studies are being carried out on the functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT). Bacterially expressed murine leukemia virus (MuLV) RT proteins (about 90% pure) are being used. These proteins include (i) wild-type RT, (ii) deltaSX, having a deletion of 127 residues immediately upstream of the RNase H domain; (iii),deltaH, missing the entire RNase H domain; and (iv) a chimeric protein having the viral polymerase domain fused to E. coli RNase H. RNase H activity is being assayed with model substrates containing sequences from the purine-rich region at the 3' end of the MuLV RNA genome. In the absence of polymerization, the results show that with wild-type RT, cleavage sites are few in number and are influenced by the position corresponding to the 3'-end of the cDNA oligonucleotide (18-nt from the end) and by the RNA sequence; the chimera makes multiple cuts and generates the same cleavage pattern as E. coli RNase H. (Experiments with a non-viral substrate also indicate that the chimera behaves like E. coli RNase H.) The deltaSX and deltaH RTs have no activity in the assay. Future efforts will include analysis of the cleavage reactions under conditions which promote viral DNA synthesis. In other work, translational control of viral gene expression is being investigated by studying readthrough suppression of the UAG codon at the MuLV gag-pol junction. Current interest is focused on the nature of the cis-acting sequences in viral mRNA which represent the suppression signal. A construct containing only two codons from the 3' end of gag, UAG, and 19 codons from the 5' end of pol has a normal level of suppressor activity. Removal of pol sequences (as little as 12 nt representing the 16-19th codons downstream of the UAG codon) completely abolishes readthrough suppression. This segment contains a run of six G's which is highly conserved in mammalian type C retroviruses. Removal of the two gag codons does not affect the efficiency of suppression. These results demonstrate that a limited region of viral mRNA, consisting of 57 nt on the 3' side of the UAG, is required for suppression. Additional mutational analysis of this region is in progress.